TECHNICAL BULLETIN. Phospho-Akt (pser 473 ) ELISA Kit for detection of human, mouse, or rat phospho-akt (pser 473 ) in cell and tissue lysates

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1 Phospho-Akt (pser 473 ) ELISA Kit for detection of human, mouse, or rat phospho-akt (pser 473 ) in cell and tissue lysates Catalog Number RAB0011 Storage Temperature 20 C TECHNICAL BULLETIN Product Description The Phospho-Akt (pser 473 ) ELISA kit is an in vitro enzyme-linked immunosorbent assay for the measurement of human, mouse, and rat phospho-akt (pser 473 ). An anti-pan-akt (pser 473 ) antibody has been coated onto a 96 well plate. Samples are pipetted into the wells and Akt present in a sample is bound to the wells by the immobilized antibody. The wells are washed and anti-akt (pser 473 ) antibody is used to detect phosphorylated Akt (pser 473 ). After washing away unbound antibody, HRP-conjugated anti-rabbit IgG is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of Akt (pser 473 ) bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm. Components 1. Capture Antibody-Coated Microplate (Item A) - RABASA: 96 wells (12 strips x 8 wells) coated with anti-pan-akt antibody x Wash Buffer Concentrate (Item B) - RABWASH5: 25 ml of 20x concentrated solution. 3. 5x Assay Diluent (Item E) - RABDIL11: 15 ml of 5x concentrated buffer. For diluting cell lysate sample, detection antibody (Item C) and HRP-conjugated Anti-Rabbit IgG Concentrate (Item D1). 4. Phospho-Akt (pser 473 )-specific Antibody Concentrate (Item C1) - RABA473C1: 2 vials of rabbit anti-akt (pser 473 ) (each vial is enough to assay half microplate). 5. HRP-conjugated Anti-Rabbit IgG Concentrate (Item D1) - RABHRP4: 25 µl of 500x HRP-conjugated Anti-rabbit IgG concentrate. 6. TMB One-Step Substrate Reagent (Item H) - RABTMB4: 12 ml of 3,3,5,5 -tetramethylbenzidine (TMB) in buffered solution. 7. Phosphorylation ELISA Stop Solution (Item I) - RABSTOP4: 8 ml of 0.2 M sulfuric acid. 8. 2x Cell Lysate Buffer (Item J) - RABCLB1: 5 ml of 2x cell lysis buffer (not including protease and phosphatase inhibitors). 9. Phopho-Akt (pser 473 ) Lyophilized Positive Control Sample (Item K) - RABA473K: 1 vial of lyophilized powder from A431 cell lysate. Reagents and Equipment Required but Not Provided. 1. Microplate reader capable of measuring absorbance at 450 nm. 2. Protease and Phosphatase inhibitors. 3. Shaker. 4. Precision pipettes to deliver 2 µl to 1 ml volumes. 5. Adjustable 1-25 ml pipettes for reagent preparation ml and 1 liter graduated cylinders. 7. Distilled or deionized water. 8. Tubes to prepare sample dilutions. Precautions and Disclaimer This product is for R&D use only, not for drug, household, or other uses. Please consult the Safety Data Sheet for information regarding hazards and safe handling practices. Preparation Instructions Sample Preparation 2x Cell Lysate Buffer should be diluted 2-fold with deionized or distilled water to yield 1x Cell Lysate Buffer (addition of protease and phosphatase inhibitors to 1x Cell Lysate Buffer is recommended prior to sample preparation).

2 2 Cell lysates - Rinse cells with PBS, making sure to remove any remaining PBS before adding the Cell Lysate Buffer. Solubilize cells at cells/ml in 1x Cell Lysate Buffer. Pipette up and down to resuspend and incubate the lysates with shaking at 2 8 C for 30 minutes. Microcentrifuge at 13,000 rpm for 10 minutes at 2 8 C and transfer the supernatants into a clean test tube. Lysates should be used immediately, or aliquoted and stored at 70 C. Avoid repeated freeze-thaw cycles. Thawed lysates should be kept on ice prior to use. For the initial experiment, it is recommend to perform serial dilution testing such as 5-fold and 50-fold dilution for the cell lysates with 1x Assay Diluent (Item E) before use. Note: The fold dilution of sample used depends on the abundance of phosphorylated proteins and should be determined empirically. More of the sample can be used if signals are too weak. If signals are too strong, the sample can be diluted further. Reagent Preparation 1. Bring all reagents and samples to room temperature (18 25 C) before use. 2. Item E, Assay Diluent should be diluted 5-fold with deionized or distilled water before use. 3. Preparation of Positive Control: Briefly spin the Positive Control vial of Item K. Add 500 µl of 1x Assay Diluent (Item E, Assay Diluent should be diluted 5-fold with deionized or distilled water before use) into Item K vial to prepare a Positive Control (P-1) Solution. Dissolve the powder thoroughly by a gentle mix (if any precipitate in the solution is found, remove by centrifugation). Pipette 300 µl of 1x Assay Diluent into each tube. Use the Positive Control (P-1) to produce a dilution series (see Figure 1). Mix each tube thoroughly before the next transfer. 1x Assay Diluent serves as the background. Figure 1. Dilution Series for Positive Control Positive Control powder µl of 1x Assay Diluent 150 µl 150 µl 150 µl P-1 P-2 P-3 P If the Wash Concentrate (20x) (Item B) contains visible crystals, warm to room temperature and mix gently until dissolved. Dilute 20 ml of Wash Buffer Concentrate into deionized or distilled water to yield 400 ml of 1x Wash Buffer. 5. Briefly spin the detection antibody (Item C) before use. Add 100 µl of 1x Assay Diluent into the vial to prepare a detection antibody concentrate. Pipette up and down to mix gently (the concentrate can be stored at 4 C for 5 days or at 70 C for one month). The anti-phospho-akt (pser 473 ) antibody should be diluted 55-fold with 1x Assay Diluent and used in Procedure, step Briefly spin the HRP-conjugated anti-rabbit IgG (Item D1) before use. Pipette up and down to mix gently. HRP-conjugated anti-rabbit IgG concentrate should be diluted 500-fold with 1x Assay Diluent. For example: Briefly spin the vial (Item D1) and pipette up to mix gently. Add 10 µl of HRPconjugated anti-rabbit IgG concentrate into a tube with 5 ml 1x of Assay Diluent to prepare a 500-fold diluted HRP-conjugated anti-rabbit IgG solution.

3 3 Storage/Stability Store the kit at 20 C Please use within 1 year from the date of shipment. Avoid repeated freeze-thaw cycles. After initial use, Wash Buffer Concentrate (Item B), Assay Diluent (Item E), TMB One-Step Substrate Reagent (Item H), Stop Solution (Item I), and Cell Lysate Buffer (Item J) should be stored at 2 8 C to avoid repeated freeze-thaw cycles. Return unused wells to the pouch containing desiccant pack, reseal along entire edge, and store at 20 C. Store Item D-1 at 2 8 C for up to one month (store at 20 C for up to 6 months, avoid repeated freeze-thaw cycles). Reconstituted Positive Control (Item K) should be stored at 70 C. Procedure 1. Bring all reagents to room temperature (18 25 C) before use. It is recommended that all samples or Positive Control should be run at least in duplicate. 2. Add 100 µl of each sample or positive control into appropriate wells. Cover well with plate holder and incubate for 2.5 hours at room temperature or overnight at 4 C with shaking. 3. Discard the solution and wash 4 times with 1x Wash Solution. Wash by filling each well with Wash Buffer (300 µl) using a multichannel pipette or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels. 4. Add 100 µl of prepared 1x detection anti-phospho- Akt (pser 473 ) antibody (see Preparation, step 5) to each well. Incubate for 1 hour at room temperature with shaking. 5. Discard the solution. Repeat the wash as in step Add 100 µl of prepared 1x HRP-conjugated Anti- Rabbit IgG (see Preparation, step 6) to each well. Incubate for 1 hour at room temperature with shaking. 7. Discard the solution. Repeat the wash as in step Add 100 µl of TMB One-Step Substrate Reagent (Item H) to each well. Incubate for 30 minutes at room temperature in the dark with shaking. 9. Add 50 µl of Stop Solution (Item I) to each well. Read at 450 nm immediately.

4 4 Results Typical Data ELISA data analysis: Average the duplicate readings for each sample or positive. Positive Control: A431 cells were treated with recombinant human EGF at 37 C for 20 minutes. Solubilize cells at cells/ml in Cell Lysate Buffer. Serial dilutions of cell lysates were analyzed in this ELISA. Please see Preparation, step 3 for detail. PDGF Stimulation of NIH3T3 Cell Lines: NIH3T3 cells were treated or untreated with PDGF for 10 minutes. Cell lysates were analyzed using this phospho ELISA kit and Western blot. ELISA Western blot References 1. Hajduch, E. et al., FEBS Lett., 492, (2001). 2. Burgering, B.M., and Coffer, P.J., Nature, 376, (1995). 3. Franke, T.F. et al., Cell, 88, (1997).

5 5 Appendix Troubleshooting Guide Problem Cause Solution Inaccurate pipetting Check pipettes Poor standard curve Ensure a brief spin of Item C and dissolve Improper standard dilution the powder thoroughly with gentle mixing. Low signal Too brief incubation times Ensure sufficient incubation time; Procedure, step 2 may change to over night Inadequate reagent volumes or Check pipettes and ensure correct improper dilution preparation Large CV Inaccurate pipetting Check pipettes Review the manual for proper wash. If using High background Plate is insufficiently washed a plate washer, check that all ports are unobstructed. Contaminated wash buffer Make fresh wash buffer Store the standard at < 20 C after Improper storage of the ELISA kit reconstitution, others at 4 C. Keep Low sensitivity substrate solution protected from light Stop solution Stop solution should be added to each well before measurement. SG,KCP,TD,KJ,MAM 02/ Sigma-Aldrich Co. LLC. All rights reserved. SIGMA-ALDRICH is a trademark of Sigma-Aldrich Co. LLC, registered in the US and other countries. Sigma brand products are sold through Sigma-Aldrich, Inc. Purchaser must determine the suitability of the product(s) for their particular use. Additional terms and conditions may apply. Please see product information on the Sigma-Aldrich website at and/or on the reverse side of the invoice or packing slip.

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